Methods and compositions for treating anxiety

ABSTRACT

Methods of treating a TRPC5 mediated disorder in a subject by administering an effective amount of a TRPC5 antagonist, such as a compound disclosed herein, are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national stage application under 35 U.S.C. §371 ofInternational Application No. PCT/US2009/052971, filed Aug. 6, 2009,published as International Publication No. WO 2010/017368 on Feb. 11,2010, which claims priority from U.S. Ser. No. 61/086,784; U.S. Ser. No.61/086,785; and U.S. Ser. No. 61/086,787, all of which were filed Aug.6, 2008, and are hereby incorporated by reference in their entities.

BACKGROUND

A variety of ion channel proteins exist to mediate ion flux acrosscellular membranes. The proper expression and function of ion channelproteins is essential for the maintenance of cell function,intracellular communication, and the like. Numerous diseases are theresult of misregulation of membrane potential or aberrant calciumhandling. Given the central importance of ion channels in modulatingmembrane potential and ion flux in cells, identification of agents thatcan promote or inhibit particular ion channels are of great interest aspotential therapeutic agents.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for treatingconditions such as pain and/or anxiety by modulating the activity of thetransient receptor potential cation channel subfamily C, member 5(TRPC5), which can exist in homomultimeric form as well asheteromultimeric form with other ion channels such as TRPC1 or TRPC3(i.e., TRPC5-TRPC1 and TRPC1-TRPC3-TRPC5). The compounds describedherein modulate the function of TRPC5 by inhibiting a TRPC5-mediated ionflux or by inhibiting the inward current, the outward current, or bothcurrents mediated by TRPC5. The inhibition of a particular current isthe ability to inhibit or reduce such current (e.g., inward and/oroutward) in an in vitro or an in vivo assay. The activation of aparticular current is the ability to activate or increase such current(e.g., inward and/or outward) in an in vitro or an in vivo assay.

In one aspect, the invention relates to a method for treating acondition for which reduced TRPC5 activity can reduce the severity ofthe condition, by administering a TRPC5 antagonist that inhibitsTRPC5-mediated current and/or TRPC5-mediated ion flux. Described ingreater detail below are TRPC5 antagonists that have measured IC₅₀'s forinhibition of TRPC5 of 10 micromolar or less, 5 micromolar or less, 2micromolar or less, 1 micromolar or less, 500 nanomolar or less, 200nanomolar or less, 100 nanomolar or less, or 10 nanomolar or less. Incertain embodiments, a TRPC5 antagonist inhibits one or both of inwardand outward TRPC5-mediated currents with an IC₅₀ of 1 micromolar orless, and more preferably with an IC₅₀ of 500 nanomolar or less, 200nanomolar or less, 100 nanomolar or less, 25 nanomolar or less, or 10nanomolar or less. In certain embodiments, the TRPC5 antagonist inhibitsat least 95% of TRPC5-mediated current or TRPC5-mediated ion flux whenadministered at 5 micromolar or less, and more preferably whenadministered at 1 micromolar or less.

In another aspect, a TRPC5 antagonist such as a compound describedherein can be used to inhibit a function of TRPC5, for example aTRPC5-mediated current and/or a TRPC5-mediated ion flux. In someembodiments, a TRPC5 antagonist can be used to inhibit a TRPC5 mediatedcurrent in vitro, for example in cells in culture. In other embodiments,a TRPC5 antagonist such as a compound described herein can be used toinhibit a TRPC5 mediated current in vivo. In certain embodiments, aTRPC5 antagonist such as a compound described herein inhibits both aninward and an outward TRPC5-mediated current.

In one aspect, the invention features a method of treating a TRPC5mediated disorder by administering an effective amount of a compound offormula (I), or a pharmaceutically acceptable salt thereof:

whereinR¹ is aryl or heteroaryl, optionally substituted with 1-5 R⁴;R² is heteroaryl, optionally substituted with 1-5 R⁵;R³ is H, C₁-C₆ alkyl, arylalkyl, or a nitrogen protecting group;each R⁴ is independently C₁-C₆ alkyl, C₁-C₆ haloalkyl, —C(O)R⁶;—C(O)OR⁶, OR⁶, —C(O)NR⁷R⁸, or 2 R⁴, taken together with the carbon towhich they are attached, form a 4-6 membered ring;each R⁵ is independently alkyl, cyclyl, heterocyclyl, aryl, orheteroaryl;each R⁶ is independently H, C₁-C₆ alkyl, or C₁-C₆ haloalkyl; andeach R⁷ and R⁸ is independently H or C₁-C₆ alkyl.

In some embodiments, R¹ is aryl, e.g., phenyl. In some embodiments, R¹is substituted by 1 R⁴. In some embodiments, R⁴ is C₁-C₆ haloalkyl. Insome embodiments, R⁴ is CF³. In some embodiments, R¹ is substituted by 2R⁴, for example, wherein R¹ is

In some embodiments, R¹ is

In some embodiments, R¹ is heteroaryl.

In some embodiments, R² is a 5 membered heteroaryl, for example, R² istetrazolyl. In some embodiments, R² is unsubstituted. In someembodiments, R² is substituted by 2 R⁵. In some embodiments, R² is

wherein each R⁵ is independently alkyl or heteroaryl. In someembodiments, R² is

In some embodiments, R² is a fused heteroaryl, for example, a 2-ringfused heteroaryl. In some embodiments, R² is

In some embodiments, R³ is H.

Exemplary compounds of formula (I) include those described in Table 1.

In another aspect, the invention features a method for treating a TRPC5mediated disorder by administering an effective amount of a compound offormula (II), or a pharmaceutically acceptable salt thereof:

whereinR¹¹ is halo, C₁-C₆ alkoxy, cycyl, heterocyclyl, aryl, or heteroaryl;optionally substituted with 1-3 R¹⁶;R¹² is OR¹⁷, SR¹⁷, or NR¹⁴R¹⁵;R¹³ is H, cycyl, heterocyclyl, aryl, or heteroaryl; optionallysubstituted with 1-3 R¹⁸;each R¹⁴ and R¹⁵ is independently H or C₁-C₆ alkyl optionallysubstituted with 1-3 R¹⁹;each R¹⁶ and R¹⁸ is independently C₁-C₆ alkyl or C₁-C₆ haloalkyl;R¹⁷ is C₁-C₆ alkyl, optionally substituted with 1-3 R¹⁹;R¹⁹ is C(O)OC₁-C₆alkyl, C(O)C₁-C₆alkyl, or OC(O)C₁-C₆alkyl.

In some embodiments, R¹¹ is cyclyl, heterocyclyl, aryl, heteroaryl, orNR¹⁴R¹⁵; optionally substituted with 1-3 R¹⁶. In some embodiments, R¹¹is cyclyl, e.g., a bridged cyclyl such as adamantyl. In someembodiments, R¹¹ is heteroaryl, for example, a nitrogen containingheteroaryl. In some embodiments, R¹¹ is aryl, e.g., phenyl. In someembodiments, phenyl is substituted by 1 R¹⁶. In some embodiments, R¹⁶ ishaloalkyl. In some embodiments, R¹¹ is C₁-C₆ alkoxy.

In some embodiments, R¹² is OR¹⁷. In some embodiments, R¹⁷ is C₁-C₆alkyl.

In some embodiments, R¹² is SR¹⁷.

In some embodiments, R¹² is NR¹⁴R¹⁵. In some embodiments, R¹² is NH₂.

In some embodiments, R¹³ is H. In some embodiments, R¹³ is cyclyl, e.g.,a bridged cyclyl such as adamantyl.

Exemplary compounds of Formula (II) include those described in Table 2.

In another aspect, the invention features a method for treating a TRPC5mediated disorder by administering an effective amount of a compound ofFormula (III), or a pharmaceutically acceptable salt thereof:

whereinR²¹ is alkyl, aryl, or heteraryl; each of which is optionallysubstituted with 1-5 R²⁵;R²² is H or C₁-C₆ alkyl;each of R^(23a) and R^(23b) is independently C₁-C₆ alkyl, or R^(23a) andR^(23b) when taken together with the nitrogen to which they are attachedform a 4-7 membered ring;each R²⁴ is independently halo, C₁-C₆ alkyl, nitro, hydroxy, alkoxy,amino, alkylamino, dialkylamino, cyano;R²⁵ is C₁-C₆ alkyl;each of X and Y is independently alkylenyl, optionally substituted with1-3 R²⁶;R²⁶ is hydroxyl or alkoxy;n0, 1, 2, 3, or 4.

In some embodiments, R²¹ is aryl, for example, phenyl.

In some embodiments, R²¹ is heteroaryl, for example, a 5 memberedheteroaryl such as thiophene.

In some embodiments, X is alkylenyl (e.g. ethylenyl), substituted with 1R²⁶. In some embodiments, R²⁶ is hydroxyl.

In some embodiments, X is ethylenyl substituted by 1 R²⁶. In someembodiments, R²⁶ is hydroxyl.

In some embodiments, X is —CH₂CHOH—

In some embodiments, R²² is H.

In some embodiments, R^(23a) and R^(23b) when taken together with thenitrogen to which they are attached form a ring

In some embodiments, Y is ethylene.

-   -   In some embodiments, the compound is a compound of formula        (IIIa), or a pharmaceutically acceptable salt thereof

Exemplary compounds of Formula (III) include those described in Table 3.

Another aspect of the invention features a pharmaceutical preparationsuitable for use in a human patient, or for veterinary use, comprisingan effective amount of any of the compounds shown herein, and one ormore pharmaceutically acceptable excipients.

DETAILED DESCRIPTION OF THE INVENTION

Cation channels such as TRPC5 modulate the flux of calcium and sodiumions across cellular membranes. Sodium and calcium influx leads to adepolarization of the cell. This increases the probability thatvoltage-gated ion channels will reach the threshold required foractivation. As a result, activation of non-selective cation channels canincrease electrical excitability and increase the frequency ofvoltage-dependent events. Voltage-dependent events include, but are notlimited to, neuronal action potentials, cardiac action potentials,smooth muscle contraction, cardiac muscle contraction, and skeletalmuscle contraction.

Calcium influx caused by the activation of non-selective cation channelssuch as TRPC5 also alters the intracellular free calcium concentration.Calcium is a ubiquitous second messenger molecule within the cell andthe alterations in intracellular calcium levels have profound effects onsignal transduction and gene expression. Thus, activation ofnon-selective cation channels such as TRPC5 can lead to changes in geneexpression and cellular phenotype. Gene expression events include, butare not limited to, production of mRNAs encoding cell surface receptors,ion channels, and kinases. These changes in gene expression can lead tohyperexcitability in that cell.

Transient receptor potential (TRP) homomeric TRPC5 ion channels arereceptor-operated, Ca²⁺-permeable channels predominantly expressed inthe neurons. TRPC5 forms homomultimeric structures such as tetramers(i.e., TRPC5 homomultimers) and heteromultimeric structures such astetramers (i.e., TRPC5-TRPC1 heteromultimers). Unless expressly statedotherwise, when the term TRPC5 is used herein, for example, whenidentifying a modulator of TRPC5 such as a TRPC5 antagonist, the termTRPC5 is used generically so as to include either or both of a TRPC5homomultimer or a heteromultimer (e.g., TRPC5-TPRC1 or TRPC5-TRPC4heteromultimer). Examples of TRPC5 in the literature include thefollowing: Nature. 2008 Jan. 3; 451 (7174):69-72; Mol Pharmacol. 2008January; 73 (1):42-9; J Biol Chem. 2007 Nov. 16; 282 (46):33868-78;Biochem Biophys Res Commun. 2008 Jan. 11; 365 (2):239-45; J Biol Chem.2006 Nov. 3; 281 (44):33487-96; Eur J Pharmacol. 2005 Mar. 14; 510(3):217-22; J Biol Chem. 2006 Feb. 24; 281 (8):4977-82; Biochem SocTrans. 2007 February; 35 (Pt 1):101-4; Handb Exp Pharmacol. 2007;(179):109-23; J Biol Chem. 2005 Mar. 25; 280 (12):10997-1006; J Physiol.2006 Jan. 15; 570 (Pt 2):219-35; and Nat Neurosci. (2003) 6: 837-45.

Modulating the function of TRPC5 proteins provides a means of modulatingcalcium homeostasis, sodium homeostasis, membrane polarization, and/orintracellular calcium levels, and compounds that can modulate TRPC5function are useful in many aspects, including, but not limited to,maintaining calcium homeostasis, modulating intracellular calciumlevels, modulating membrane polarization, and treating or preventingdiseases, disorders, or conditions associated with calcium and/or sodiumhomeostasis or dyshomeostasis.

In certain aspects, the present invention provides methods for treatingor ameliorating the effects of diseases and conditions using a compounddescribes herein that inhibits a TRPC5-mediated current and/or aTRPC5-mediated ion flux with an IC₅₀ of less than 10 micromolar.

In certain embodiments, a TRPC5 antagonist inhibits an inward and/oroutward TRPC5 mediated current with an IC₅₀ of less than 10 micromolar(e.g., less than 1 micromolar). In certain embodiments, a TRPC5antagonist inhibits TRPC5 mediated ion flux with an IC₅₀ of less than 10micromolar. The IC₅₀ can be calculated, for example, in an in vitroassay. For example, IC₅₀ can be calculated using electrophysiologicaldeterminations of current, such as standard patch clamp analysis. IC₅₀can also be evaluated using changes in concentration or flux of ionindicators, such as the calcium flux methods described herein.

In some embodiments, a TRPC5 antagonist (e.g., a compound describedherein) is chosen because it inhibits a TRPC5 function with an IC₅₀ lessthan or equal to 1 uM, or even less than or equal to 700, 600, 500, 400,300, 250, 200, or 100 nM. In other embodiments, the small molecule ischosen because it inhibits a TRPC5 function with an IC₅₀ less than orequal to 75 nM, less than or equal to 50 nM, or even less than or equalto 25, 10, 5, or 1 nM.

In some embodiments, a TRPC5 antagonist (e.g., a compound describedherein) is chosen based on the rate of inhibition of a TRPC5 function.In one embodiment, the compound inhibits a TRPC5 function in less than 5minutes, preferably less than 4, 3, or 2 minutes. In another embodiment,the compound inhibits a TRPC5 function in less than about 1 minute. Inyet another embodiment, the compound inhibits a TRPC5 function in lessthan about 30 seconds.

In certain embodiments of any of the foregoing, inhibition of a TRPC5function means that a function, for example a TRPC5 mediated current, isdecreased by greater than 50% in the presence of an effective amount ofa compound in comparison to in the absence of the compound or incomparison to an ineffective amount of a compound. In certain otherembodiments, the inhibition of a TRPC5 function means that a function,for example a TRPC5 mediated current or TRPC5 mediated ion flux, isdecreased by at least 50%, 60%, 70%, 75%, 80%, 85%, or 90% in thepresence of an effective amount of a compound in comparison to in theabsence of the compound. In still other embodiments, the inhibition of aTRPC5 function means that a function, for example a TRPC5 mediatedcurrent, is decreased by at least 92%, 95%, 97%, 98%, 99%, or 100% inthe presence of an effective amount of a compound in comparison to inthe absence of the compound.

In certain embodiments, a TRPC5 antagonist (e.g., a compound describedherein) is chosen for use because it is more potent against TRPC5 thanfor other TRP ion channels, (including, but not limited to, TRPV5,TRPV3, TRPV4, TRPM8, TRPA1, TRPC3, TRPV6, TRPC7, TRPV6, and TRPV1). Forexample, the TRPC5 antagonist can be 10-fold, and more preferably atleast 20, 40, 50, 60, 70, 80, or at least 100-fold or at least 1000-foldmore potent against TRPC5 than for the other TRP channel.

The TRPC5 antagonist can also be more potent against TRPC5 than againstNaV1.2, Cav1.2, Cav3.1, hERG, and/or the mitochondrial uniporter.

In certain embodiments, a TRPC5 antagonist (e.g., a compound describedherein) has a therapeutic index (T.I.) for treating the condition withthe compound of 3 or greater, and more preferably has a T.I. of at least5, 10, 25, 50 or 100.

Exemplary compounds are provided in Tables 1-3, below:

TABLE 1

TABLE 2

TABLE 3

Compounds of any of the above structures may be used to inhibit afunction of a TRPC5 channel in vitro or in vivo.

DEFINITIONS

The term “acyl” is art-recognized and refers to a group represented bythe general formula hydrocarbylC(O)—, preferably alkylC(O)—.

The term “acylamino” is art-recognized and refers to a moiety that canbe represented by the general formula:

wherein R⁹ is as defined above, and R′¹¹ represents a hydrogen, analkyl, an alkenyl or —(CH₂)m-R⁸, where m and R⁸ are as defined above.

The term “aliphatic group” refers to a straight-chain, branched-chain,or cyclic aliphatic hydrocarbon group and includes saturated andunsaturated aliphatic groups, such as an alkyl group, an alkenyl group,and an alkynyl group.

The term “alkenyl”, as used herein, refers to an aliphatic groupcontaining at least one double bond.

The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group,as defined below, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C₁-C₃₀ for straight chains, C₃-C₃₀ for branchedchains), and more preferably 20 or fewer, and most preferably 10 orfewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms intheir ring structure, and more preferably have 5, 6 or 7 carbons in thering structure.

The term “alkynyl”, as used herein, refers to an aliphatic groupcontaining at least one triple bond.

The term “alkylthio” refers to an hydrocarbyl having a sulfur radicalattached thereto. In preferred embodiments, the “alkylthio” moiety isrepresented by one of —S-alkyl, —S-alkenyl, —S-alkynyl. Representativealkylthio groups include methylthio, ethylthio, and the like.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

wherein R⁹, R¹⁰ and R′¹⁰ each independently represent a hydrogen, analkyl, an alkenyl, —(CH₂)_(m)—R⁸, or R⁹ and R¹⁰ taken together with theN atom to which they are attached complete a heterocycle having from 4to 8 atoms in the ring structure; R⁸ represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8.

The term “amido” refers to a moiety that can be represented by thegeneral formula:

wherein R⁹, R¹⁰ are as defined above.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group (e.g., an aromatic or heteroaromatic group).

The term “aryl” as used herein includes 5-, 6-, and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,polycyclyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido,phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether,alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl,aromatic or heteroaromatic moieties, —CF₃, —CN, or the like. The term“aryl” also includes polycyclic ring systems having two or more cyclicrings in which two or more carbons are common to two adjoining rings(the rings are “fused rings”) wherein at least one of the rings isaromatic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The term “carbocycle or cycyl”, as used herein, refers to an aromatic ornon-aromatic ring in which each atom of the ring is carbon.

The term “carbonyl” refers to moieties represented by the generalformula:

wherein X is a bond or represents an oxygen or a sulfur, and R¹¹represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R⁸ or apharmaceutically acceptable salt, R′¹¹ represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R⁸, where m and R⁸ are as defined above. WhereX is an oxygen and R¹¹ or R′¹¹ is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R¹¹ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR¹¹ is a hydrogen, the formula represents a “carboxylic acid”. Where Xis an oxygen, and R′¹¹ is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiocarbonyl” group. Where X is asulfur and R¹¹ or R′¹¹ is not hydrogen, the formula represents a“thioester.” Where X is a sulfur and R¹¹ is hydrogen, the formularepresents a “thiocarboxylic acid.” Where X is a sulfur and R¹¹′ ishydrogen, the formula represents a “thiolformate.” On the other hand,where X is a bond, and R¹¹ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R¹¹ is hydrogen, the aboveformula represents an “aldehyde” group.

The term “ester”, as used herein, refers to a group —C(O)OR⁹ wherein R⁹represents a hydrocarbyl group.

The terms “halo” and “halogen” as used herein means halogen and includeschloro, fluoro, bromo, and iodo.

The terms “hetaralkyl” and “heteroaralkyl”, as used herein, refers to analkyl group substituted with a hetaryl group.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles, with each group having, e.g., 5-7 ring members. Theterm “heterocyclyl” or “heterocyclic group” includes “heteroaryl” and“saturated or partially saturated heterocyclyl” structures. The term“heteroaryl” refers to an aromatic 5-8 membered monocyclic, 8-12membered bicyclic, or 11-14 membered tricyclic ring system having 1-3heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively). Any ring atom can besubstituted (e.g., by one or more substituents). The term “saturated orpartially saturated heterocyclyl” refers to a non-aromatic cylicstructure that includes at least one heteroatom. Heterocyclyl groupsinclude, for example, thiophene, thianthrene, furan, pyran,isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole,pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine,pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactamssuch as azetidinones and pyrrolidinones, sultams, sultones, and thelike. The heterocyclic ring can be substituted at one or more positionswith such substituents as described above, as for example, halogen,alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl,carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, aheterocyclyl, an aromatic or heteroaromatic moiety, —CF₃, —CN, or thelike.

The term “heterocyclylalkyl”, as used herein, refers to an alkyl groupsubstituted with a heterocycle group.

The term “hydrocarbyl”, as used herein, refers to a group that is bondedthrough a carbon atom that does not have a ═O or ═S substituent, andtypically has at least one carbon-hydrogen bond and a primarily carbonbackbone, but may optionally include heteroatoms. Thus, groups likemethyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered to behydrocarbyl for the purposes of this application, but substituents suchas acetyl (which has a ═O substituent on the linking carbon) and ethoxy(which is linked through oxygen, not carbon) are not. Hydrocarbyl groupsinclude, but are not limited to aryl, heteroaryl, carbocycle,heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.

As used herein, the term “nitro” means —NO₂; the term “halogen” or“halo” designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH;the term “hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

Exemplary monocyclic rings include furan, thiophene, pyrrole, pyrroline,pyrrolodine, oxazole, thiazole, imidazole, imidazoline, pyrazole,pyrazoline, pyrazolidine, isoxazole, isothiazole, oxadiazole, triazole,thiadiazole, pyran, pyridine, piperidine, dioxane, morpholine, dithiane,thiomorpholine, pyridazine, pyrimidine, pyrazine, piperazine, triazine,and trithiane.

Exemplary bicyclic rings include indolizinyl, indolyl, isoindolyl,indolinyl, benzofuranyl, benzothiophenyl, indazolyl, benzimidazolyl,benthiazolyl, purinyl, quinolizinyl, quinolinyl, isoquinolinyl,cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl,pteridinyl, indenyl, naphthalenyl, azulenyl, imidazopyridazionyl,pyrazolopyrimidinedionyl, or pyrrolopyrimidinedionyl moieties.

Exemplary tricyclic rings include carbazole, acridine, phenazine,phenothiazine, phenoxazine, fluorine, and anthracene.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991).

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include any substituents described herein,for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, aphosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine,an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, asulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, aheterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. For instance, the substituents of a substituted alkyl mayinclude substituted and unsubstituted forms of amino, azido, imino,amido, phosphoryl (including phosphonate and phosphinate), sulfonyl(including sulfate, sulfonamido, sulfamoyl and sulfonate), and silylgroups, as well as ethers, alkylthios, carbonyls (including ketones,aldehydes, carboxylates, and esters), —CF₃, —CN and the like. Exemplarysubstituted alkyls are described below. Cycloalkyls can be furthersubstituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls,carbonyl-substituted alkyls, —CF₃, —CN, and the like. Analogoussubstitutions can be made to alkenyl and alkynyl groups to produce, forexample, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls,iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

The term “sulfate” refers to a moiety that can be represented by thegeneral formula:

in which R⁴¹ is as defined above.

The term “sulfonamido” refers to a moiety that can be represented by thegeneral formula:

in which R⁹ and R′¹¹ are as defined above.

The term “sulfonate” refers to a moiety that can be represented by thegeneral formula:

in which R⁴¹ is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms “sulfoxido” or “sulfinyl”, as used herein, refers to a moietythat can be represented by the general formula —S(═O)—R⁴⁴, in which R⁴⁴is selected from the group consisting of hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

The term “thioester”, as used herein, refers to a group —C(O)SR⁹ or—SC(O)R⁹ wherein R⁹ represents a hydrocarbyl.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

Certain compounds disclosed herein may exist in particular geometric orstereoisomeric forms. The present invention contemplates all suchcompounds, including cis- and trans-isomers, R- and S-enantiomers,diastereomers, (d)-isomers, (l)-isomers, the racemic mixtures thereof,and other mixtures thereof, as falling within the scope of theinvention. For example, if one chiral center is present in a molecule,the invention includes racemic mixtures, enantiomerically enrichedmixtures, and substantially enantiomerically pure compounds. Thecomposition can contain, e.g., more than 50%, more than 60%, more than70%, more than 80%, more than 90%, more than 95%, or more than 99% of asingle enantiomer.

The “enantiomeric excess” or “% enantiomeric excess” of a compositioncan be calculated using the equation shown below. In the example shownbelow a composition contains 90% of one enantiomer, e.g., the Senantiomer, and 10% of the other enantiomer, i.e., the R enantiomer.ee=(90−10)/100=80%.Thus, a composition containing 90% of one enantiomer and 10% of theother enantiomer is said to have an enantiomeric excess of 80%.Compositions have ee's of at least 90%, 95%, 98%, 99%, 99.5%, and 99.9%are included within the scope of the invention.

Methods of preparing substantially isomerically pure compounds are knownin the art. If, for instance, a particular enantiomer of a compounddisclosed herein is desired, it may be prepared by asymmetric synthesis,or by derivation with a chiral auxiliary, where the resultingdiastereomeric mixture is separated and the auxiliary group cleaved toprovide the pure desired enantiomers. Alternatively, where the moleculecontains a basic functional group, such as amino, or an acidicfunctional group, such as carboxyl, diastereomeric salts may be formedwith an appropriate optically active acid or base, followed byresolution of the diastereomers thus formed by fractionalcrystallization or chromatographic means well known in the art, andsubsequent recovery of the pure enantiomers. Alternatively,enantiomerically enriched mixtures and pure enantiomeric compounds canbe prepared by using synthetic intermediates that are enantiomericallypure in combination with reactions that either leave the stereochemistryat a chiral center unchanged or result in its complete inversion.Techniques for inverting or leaving unchanged a particular stereocenter,and those for resolving mixtures of stereoisomers are well known in theart, and it is well within the ability of one of skill in the art tochoose an appropriate method for a particular situation. See, generally,Furniss et al. (eds.), Vogel's Encyclopedia of Practical OrganicChemistry 5^(th) Ed., Longman Scientific and Technical Ltd., Essex,1991, pp. 809-816; and Heller, Acc. Chem. Res. 23: 128 (1990).

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the samegeneral properties thereof (e.g., the ability to inhibit TRPC5activity), wherein one or more simple variations of substituents aremade which do not adversely affect the efficacy of the compound. Ingeneral, the compounds disclosed herein may be prepared by the methodsillustrated in the general reaction schemes as, for example, describedbelow, or by modifications thereof, using readily available startingmaterials, reagents and conventional synthesis procedures. In thesereactions, it is also possible to make use of variants which are inthemselves known, but are not mentioned here.

The compounds described herein may also contain unnatural proportions ofatomic isotopes at one or more of the atoms that constitute suchcompounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as for example tritium (³H), iodine-125(¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compoundsdisclosed herein, whether radioactive or not, are intended to beencompassed within the scope of the present invention. For example,deuterated compounds and compounds incorporating ¹³C are intended to beencompassed within the scope of the invention.

Certain compounds disclosed herein can exist in unsolvated forms as wellas solvated forms, including hydrated forms. In general, the solvatedforms are equivalent to unsolvated forms and are encompassed within thescope of the present invention. Certain compounds disclosed herein mayexist in multiple crystalline or amorphous forms. In general, allphysical forms are equivalent for the uses contemplated by the presentinvention and are intended to be within the scope of the presentinvention.

As set out above, certain embodiments of the present compounds maycontain a basic functional group, such as amino or alkylamino, and arethus capable of forming pharmaceutically acceptable salts withpharmaceutically acceptable acids. The term “pharmaceutically acceptablesalts” in this respect, refers to the relatively non-toxic, inorganicand organic acid addition salts of compounds disclosed herein. Thesesalts can be prepared in situ during the final isolation andpurification of the compounds of the invention, or by separatelyreacting a purified compound of the invention in its free base form witha suitable organic or inorganic acid, and isolating the salt thusformed. Representative salts include the hydrobromide, hydrochloride,sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate,palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate,glucoheptonate, lactobionate, and laurylsulphonate salts and the like.(See, for example, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm.Sci. 66:1-19.)

In other cases, the compounds disclosed herein may contain one or moreacidic functional groups and, thus, are capable of formingpharmaceutically acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds disclosed herein. These salts can likewise beprepared in situ during the final isolation and purification of thecompounds, or by separately reacting the purified compound in its freeacid form with a suitable base, such as the hydroxide, carbonate orbicarbonate of a pharmaceutically acceptable metal cation, with ammonia,or with a pharmaceutically acceptable organic primary, secondary ortertiary amine. Representative alkali or alkaline earth salts includethe lithium, sodium, potassium, calcium, magnesium, and aluminum saltsand the like. Representative organic amines useful for the formation ofbase addition salts include ethylamine, diethylamine, ethylenediamine,ethanolamine, diethanolamine, piperazine and the like. (See, forexample, Berge et al., supra)

The terms “antagonist” and “inhibitor” are used interchangeably to referto an agent that decreases or suppresses a biological activity, such asto repress an activity of an ion channel, such as TRPC5. TRPC5inhibitors include inhibitors having any combination of the structuraland/or functional properties disclosed herein.

An “effective amount” of, e.g., a TRPC5 antagonist, with respect to thesubject methods of inhibition or treatment, refers to an amount of theantagonist in a preparation which, when applied as part of a desireddosage regimen brings about a desired clinical or functional result.Without being bound by theory, an effective amount of a TRPC5 antagonistfor use in the methods of the present invention, includes an amount of aTRPC5 antagonist effective to decrease one or more in vitro or in vivofunctions of a TRPC5 channel. Exemplary functions include, but are notlimited to, membrane polarization (e.g., an antagonist may promotehyperpolarization of a cell), ion flux, ion concentration in a cell,outward current, and inward current. Compounds that antagonize TRPC5function include compounds that antagonize an in vitro or in vivofunctional activity of TRPC5. When a particular functional activity isonly readily observable in an in vitro assay, the ability of a compoundto inhibit TRPC5 function in that in vitro assay serves as a reasonableproxy for the activity of that compound. In certain embodiments, aneffective amount is an amount sufficient to inhibit a TRPC5-mediatedcurrent and/or the amount sufficient to inhibit TRPC5 mediated ion flux.

The term “hydrate” as used herein, refers to a compound formed by theunion of water with the parent compound.

The term “oxidative metabolite” is intended to encompass compounds thatare produced by metabolism of the parent compound under normalphysiological conditions. Specifically, an oxidative metabolite isformed by oxidation of the parent compound during metabolism. Forexample, a thioether group may be oxidized to the correspondingsulfoxide or sulfone.

The term “preventing,” when used in relation to a condition, such as alocal recurrence (e.g., pain), a disease such as cancer, a syndromecomplex such as heart failure or any other medical condition, is wellunderstood in the art, and includes administration of a compositionwhich reduces the frequency of, or delays the onset of, symptoms of amedical condition in a subject relative to a subject which does notreceive the composition. Thus, prevention of cancer includes, forexample, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount. Prevention of an infection includes, for example,reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

The term “prodrug” is intended to encompass compounds that, underphysiological conditions, are converted into the therapeutically activeagents of the present invention. A common method for making a prodrug isto include selected moieties that are hydrolyzed under physiologicalconditions to reveal the desired molecule. In other embodiments, theprodrug is converted by an enzymatic activity in the host animal.

The term “solvate” as used herein, refers to a compound formed bysolvation (e.g., a compound formed by the combination of solventmolecules with molecules or ions of the solute).

The terms “TRPC5”, “TRPC5 protein”, and “TRPC5 channel” are usedinterchangeably throughout the application. Unless expressly stated, theterm TRPC5 includes homomultimeric structures (e.g., homomultimericTRPC5) and heteromultimeric structures (e.g., heteromultimericTRPC5-TRPC1).

Diseases, Disorders, or Conditions Related to TRPC5 Function

In certain embodiments, the invention provides methods and compositionsfor antagonizing a function of a TRPC5 channel in vitro or in vivo.Exemplary functions include, but are not limited to, TRPC5-mediatedcurrent. In certain embodiments, the invention provides methods fortreating a disease or disorder or condition by administering an agentthat modulates the level and/or activity of a TRPC5 protein. In otherembodiments, the compound selectively inhibits the expression leveland/or activity of a TRPC5 protein. In other words, in certainembodiment, the compound inhibits the activity of a TRPC5 proteinpreferentially in comparison to the activity of one or more other ionchannels.

Treatment of Anxiety and Fear-Related Disorders

In certain embodiments, the TRPC5 antagonist can be used for preventingor treating anxiety and fear-related disorders (see, e.g., Riccio et al.(2009) Cell 137:761-72). Examples of such disorders includepost-traumatic stress disorder, panic disorder, agoraphobia, socialphobias, generalized anxiety disorder, panic disorder, social anxietydisorder, obsessive-compulsive disorder, and separation anxiety.

Memory, Motion, and Mood Disorders

A TRPC5 antagonist (e.g., a compound described herein) is also usefulfor the treatment of Parkinson's disease, epilepsy, memory disorders,stroke, seizure, and mood disorders. Mood disorders include depression(e.g., major depression, psychiatric depression, dysthymia, andpostpartum depression) and bipolar disorder (e.g., bipolar I, bipolarII, and cyclothymia). Memory disorders are conditions associated withany memory loss and may result from Alzheimer's disease, amnesia,aphasia, atherosclerosis, brain injury or disorder, brain tumor, chronicfatigue syndrome, Creutzfedt-Jacob disease, dissociative amnesia,depression, fuge amnesia, Huntington's disease, learning disorders,sleeping disorders, multiple personality disorder, pain, post-traumaticstress disorder, schizophrenia, sports injuries, stroke, andWernicke-Korsakoff syndrome.

Treatment of Pain, Sensitivity to Pain and Touch, or Pain-RelatedDiseases or Disorders

In certain embodiments, the TRPC5 inhibitor is used to treat orameliorate pain. Exemplary classes of pain that can be treated using aTRPC5 inhibitor include, but are not limited to nociceptive pain,inflammatory pain, and neuropathic pain. The pain can be chronic oracute.

TRPC5 inhibitors may be particularly useful in the treatment of painassociated with cancer, osteoarthritis, rheumatoid arthritis,post-herpetic neuralgia, burns, and other indications detailed above. Tofurther illustrate, additional exemplary indications for which compoundsdisclosed herein can be used include oral pain, pelvic pain, Fabry'sdisease, complex regional pain syndrome, pancreatitis, and fibromyalgiasyndrome.

The compounds disclosed herein may also be used in connection withprevention or treatment of sensitivity to pain and touch. Pain orsensitivity to pain and touch may be indicated in a variety of diseases,disorders or conditions, including, but not limited to, diabeticneuropathy, breast pain, psoriasis, eczema, dermatitis, burn,post-herpetic neuralgia (shingles), nociceptive pain, peripheralneuropathic and central neuropathic pain, chronic pain, cancer and tumorpain, spinal cord injury, crush injury and trauma induced pain,migraine, cerebrovascular and vascular pain, sickle cell disease pain,rheumatoid arthritis pain, musculoskeletal pain including treating signsand symptoms of osteoarthritis and rheumatoid arthritis, orofacial andfacial pain, including dental, temperomandibular disorder, and cancerrelated, lower back or pelvic pain, surgical incision related pain,inflammatory and non-inflammatory pain, visceral pain, psychogenic painand soft tissue inflammatory pain, fibromyalgia-related pain, and reflexsympathetic dystrophy, and pain resulting from kidney stones or urinarytract infection.

Oral pain is a particular category of pain that may be treated using theTRPC5 inhibitors disclosed herein. The term “oral pain” refers to anypain in the mouth, throat, lips, gums, teeth, tongue, or jaw. The termis used regardless of the cause of the pain and regardless of whetherthe oral pain is a primary or secondary symptom of a particular disease,injury, or condition.

In certain embodiments, oral pain is caused by ulcers, sores, or otherlesions in the mouth. For example, oral pain may be caused by ulcers,sores, or other lesions on the tongue, gums, lips, throat, or othertissues of the mouth. Alternatively or additionally, oral pain may becaused by inflammation of the throat, tongue, gums, lips, or othertissues of the mouth. Inflammation may accompany ulcers or otherlesions, or inflammation may occur prior to or in the absence offormation of ulcers or other lesions.

The foregoing are merely exemplary of diseases and conditions that causeor lead to inflammation, lesions, ulcers, or other sources of oral pain.In other embodiments, the oral pain is due to an injury to the mouth,jaw, lips, gums, or teeth. In other embodiments, the oral pain is due tooral surgery, for example, surgery for cancer, tooth extraction, or jawremodeling. Other conditions that may lead to oral ulcers, and thus oralpain, include, but are not limited to chickpox, herpes zoster,infectious mononucleosis, syphilis, tuberculosis, acute necrotizinggingivitis, and burning mouth syndrome.

Fibromyalgia (FMS; fibromyalgia syndrome) is a widespreadmusculoskeletal pain and fatigue disorder. Fibromyalgia is characterizedby pain in the muscles, ligaments, and tendons. The condition affectsmore women than men, and occurs in people of all ages. Overall, FMS isestimated to afflict 3-6% of the population. Patients have described thepain associated with fibromylagia as deep muscular aching, throbbing,shooting, and stabbing. The pain sometimes includes an intense burningsensation. The pain and stiffness are often worse in the morning orafter repetitive use of a particular muscle group.

Additionally, varying levels of fatigue ranging from mild toincapacitating are often associated with fibromylagia. Other symptoms offibromylagia include gastrointestinal symptoms. Irritable bowel syndromeand IBS-like symptoms such as constipation, diarrhea, frequent abdominalpain, abdominal gas, and nausea occur in roughly 40 to 70% of FMSpatients. Acid reflux or gastroesophogeal reflux disease (GERD) occursat a similar frequency.

Complex Regional Pain Syndrome (CRPS; also known as chronic regionalpain syndrome) is a chronic pain condition. CRPS was formerly known asreflex sympathetic dystrophy (RSD). CRPS is a chronic, painful, andprogressive neurological condition that affects skin, muscles, joints,and bones. The syndrome usually develops in an injured limb, such as abroken leg or following surgery. However, many cases involve only aminor injury, such as a sprain, and sometimes no precipitating injuriousevent can be identified. CRPS involves continuous, intense pain that isdisproportionate to the severity of the injury. The pain worsens, ratherthan improves, over time.

Although CRPS can affect a variety of regions of the body, it most oftenaffects the arms, legs, hands, or feet. Often the pain begins in oneportion of a limb, but spreads over time to include the entire limb oreven to include a different limb. Typical features include dramaticchanges in the color and temperature of the skin over the affected limbor body part, accompanied by intense burning pain, skin sensitivity,sweating, and swelling.

The compounds disclosed herein can also be used to treat endometriosisand the pain associated therewith.

Respiratory Disorders

The compounds described herein are useful for the treatment orprevention of respiratory conditions. Such conditions affect the lung,pleural cavity, bronchial tubes, trachea, upper respiratory tract aswell as the nerves and muscles involved in breathing. Respiratorydiseases that may be treated with the compounds described herein includeobstructive diseases such as chronic obstructive pulmonary disease(COPD), chronic cough, emphysema, chronic bronchitis, asthma (includingasthma caused by industrial irritants), cystic fibrosis, bronchiectasis,bronchiolitis, allergic bronchopulmonary aspergillosis, andtuberculosis; restrictive lung disease including asbestosis, radiationfibrosis, hypersensitivity pneumonitis, infant respiratory distresssyndrome, idiopathic pulmonary fibrosis, idiopathic pulmonary fibrosis,idiopathic interstial pneumonia sarcoidosis, eosinophilic pneumonia,lymphangioleiomyomatosis, pulmonary Langerhan's cell histiocytosis, andpulmonary alveolar proteinosis; respiratory tract infections includingupper respiratory tract infections (e.g., common cold, sinusitis,tonsillitis, pharyngitis and laryngitis) and lower respiratory tractinfections (e.g., pneumonia); respiratory tumors whether malignant(e.g., small cell lung cancer, non-small cell lung cancer,adenocarcinoma, squamous cell carcinoma, large cell undifferentiatedcarcinoma, carcinoid, mesothelioma, metastatic cancer of the lung,metastatic germ cell cancer, metastatic renal cell carcinoma) or benign(e.g., pulmonary hamartoma, congenital malformations such as pulmonarysequestration and congenital cystic adenomatoid malformation (CCAM));pleural cavity diseases (e.g., empyema and mesothelioma); and pulmonaryvascular diseases (e.g, pulmonary embolism such as thromboembolism, andair embolism (iatrogenic), pulmonary arterial hypertension, pulmonaryedema, pulmonary hemorrhage, inflammation and damage to capillaries inthe lung resulting in blood leaking into the alveoli. Other conditionsthat may be treated include disorders that affect breathing mechanics(e.g., obstructive sleep apnea, central sleep apnea, amyotrophic lateralsclerosis, Guillan-Barre syndrome, and myasthenia gravis). The presentcompounds can also be useful for treating, reducing, or preventing oneor more symptoms associated with respiratory conditions including, forexample, shortness of breath or dyspnea, cough (with or without theproduction of sputum), cough associated with asthma, cough associatedwith influenza, coughing blood (haemoptysis), chest pain includingpleuritic chest pain, noisy breathing, wheezing, and cyanosis.

Neurological or Neurodegenerative Diseases and Disorders

Neurodegenerative diseases and disorders include but are not limited toAlzheimer's disease (AD), Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis (ALS), and other brain disorders caused bytrauma or other insults including aging.

Mechanisms associated with calcium signaling may be altered in manyneurodegenerative diseases and in disorders resulting from brain injury.For example, fibroblasts or T-lymphocytes from patients with AD haveconsistently displayed an increase in Ca²⁺ release from intracellularstores compared to controls (Ito et al. (1994) Proc. Natl. Acad. Sci.U.S.A. 91:534-538; Gibson et al. (1996) Biochem. Biophys. ACTA1316:71-77; Etchenberrigaray et al. (1998) Neurobiology of Disease,5:37-45). Consistent with these observations, mutations in presenilingenes (PS1 or PS2) associated with familial AD (FAD) have been shown toincrease InsP3-mediated Ca²⁺ release from internal stores (Guo et al.(1996) Neuro Report, 8:379-383; Leissring et al. (1999) J.Neurochemistry, 72:1061-1068; Leissring et al. (1999) J. Biol. Chem. 274(46):32535-32538; Leissring et al. (2000) J. Cell Biol. 149 (4):793-797;Leissring et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97(15):8590-8593). Furthermore, mutations in PS1 or PS2 associated with anincrease in amyloidogenic amyloid β peptide generation in AD arereported to be associated with a decrease in intracellular calcium level(Yoo et al. (2000) Neuron, 27 (3):561-572).

Experimental traumatic brain injury has been shown to initiate massivedisturbances in Ca²⁺ concentrations in the brain that may contribute tofurther neuronal damage. Intracellular Ca²⁺ may be elevated by manydifferent ion channels. It has been further shown that channel blockersmay be beneficial in the treatment of neurological motor dysfunctionwhen administered in the acute posttraumatic period (Cheney et al.(2000) J. Neurotrauma, 17 (1):83-91).

Incontinence

Incontinence is a significant social and medical problem affecting bothmen and women. Incontinence has many causes including, but not limitedto, age, pregnancy, radiation exposure, surgery, injury, cancer,enlargement of the prostatic, prostatic hyperplasia, and diseases of thebladder or musculature that supports the urethra. The inventioncontemplates methods for treating incontinence due to any of theforegoing, as well as incontinence of unknown cause or continence due toanxiety, stress, or depression.

In certain embodiments, the compounds disclosed herein are used toreduce bladder hyperactivity by decreasing the activity of the neuronsthat innervate the bladder. In certain embodiments, incontinence isaccompanied by pain. For example, incontinence incident to bladdercystitis or incontinence incident to an injury may be accompanied bypain. When incontinence is accompanied by pain, the compound may beadministered to treat both incontinence and to reduce pain.

the sensation of cool, cold and decreased temperatures that oftenaccompany pain.

Combination Therapy

The present invention provides TRPC5 inhibitors for use in vitro and invivo. The present invention also provides compositions andpharmaceutical compositions comprising particular classes of compoundsthat inhibit TRPC5 activity. In certain embodiments, the subject TRPC5inhibitors are selective. In other words, in certain embodiments, thecompound inhibits TRPC5 activity preferentially over the activity ofother ion channels. In certain embodiments, the compound inhibits TRPC5activity preferentially over TRPV1, TRPV2, TRPV3, TRPV4, TRPC3, TRPC6,TRPC7, TRPA1, and/or TRPM8 activity. In certain other embodiments, thecompound is selected because it cross reacts with one or more other TRPchannels involved with pain. For example, in certain embodiments, thecompound inhibits the activity of TRPC5 and also inhibits the activityof one or more of TRPV1, TRPV2, TRPV3, TRPV4, TRPC3, TRPC6, TRPC7,TRPA1, and TRPM8.

TRPC5 antagonists can be used alone or in combination with otherpharmaceutically active agents. Examples of such other pharmaceuticallyactive agents include, but are not limited to, anti-depressants,anti-anxiety agents, anti-epileptic agents, anti-inflammatory agents(e.g., NSAIDS, bradykinin receptor antagonists, hormones and autacoidssuch as corticosteroids), anti-acne agents (e.g., retinoids),anti-wrinkle agents, anti-scarring agents, anti-incontinence agents(such as M1-receptor antagonists) anti-emetics (such as NK1antagonists), anti-psoriatic agents, antacids, anti-proliferative agents(e.g., anti-eczema agents, anti-cancer), anti-fungal agents, anti-viralagents, anti-septic agents (e.g., antibacterials), local anaesthetics,anti-migraine agents, keratolytic agents, hair growth stimulants, hairgrowth inhibitors, and other agents used for the treatment of skindiseases or conditions. Certain active agents belong to more than onecategory.

In certain embodiments, a compound of the invention is conjointlyadministered with an analgesic. Suitable analgesics include, but are notlimited to, opioids, glucocorticosteroids, non-steroidalanti-inflammatories, naphthylalkanones, oxicams, para-aminophenolderivatives, propionic acids, propionic acid derivatives, salicylates,fenamates, fenamate derivatives, pyrozoles, and pyrozole derivatives.Examples of such analgesic compounds include, but are not limited to,codeine, hydrocodone, hydromorphone, levorpharnol, morphine, oxycodone,oxymorphone, butorphanol, dezocine, nalbuphine, pentazocine, etodolac,indomethacin, sulindac, tolmetin, nabumetone, piroxicam, acetaminophen,fenoprofen, flurbiprofen, ibuprofen, ketoprofen, naproxen, diclofenac,oxaprozin, aspirin, diflunisal, meclofenamic acid, mefanamic acid,prednisolone, and dexamethasone. Preferred analgesics are non-steroidalanti-inflammatories and opioids (preferably morphine).

In some embodiments, the compounds disclosed herein can be administeredin conjunction with a therapeutic whose administration causes pain. Forexample, a compound described herein can be administered in conjunctionwith an anesthetic, to reduce the pain caused by the administration ofthe anaesthetic. A compound described herein can also be administered inconjunction with a chemotherapeutic agent, to reduce the pain caused byadministration of the chemotherapeutic agent.

In certain embodiments, a compound of the invention is conjointlyadministered with a non-steroidal anti-inflammatory. Suitablenon-steroidal anti-inflammatory compounds include, but are not limitedto, piroxicam, diclofenac, etodolac, indomethacin, ketoralac, oxaprozin,tolmetin, naproxen, flubiprofen, fenoprofen, ketoprofen, ibuprofen,mefenamic acid, sulindac, apazone, phenylbutazone, aspirin, celecoxiband rofecoxib.

Pharmaceutical Compositions

While it is possible for a compound disclosed herein to be administeredalone, it is preferable to administer the compound as a pharmaceuticalformulation, where the compound is combined with one or morepharmaceutically acceptable excipients or carriers. The compoundsdisclosed herein may be formulated for administration in any convenientway for use in human or veterinary medicine. In certain embodiments, thecompound included in the pharmaceutical preparation may be activeitself, or may be a prodrug, e.g., capable of being converted to anactive compound in a physiological setting.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

Examples of pharmaceutically acceptable carriers include: (1) sugars,such as lactose, glucose and sucrose; (2) starches, such as corn starchand potato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) phosphate buffer solutions; (21)cyclodextrins such as Captisol®; and (22) other non-toxic compatiblesubstances employed in pharmaceutical formulations.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Solid dosage forms (e.g., capsules, tablets, pills, dragees, powders,granules and the like) can include one or more pharmaceuticallyacceptable carriers, such as sodium citrate or dicalcium phosphate,and/or any of the following: (1) fillers or extenders, such as starches,lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders,such as, for example, carboxymethylcellulose, alginates, gelatin,polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such asglycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate; (5) solution retarding agents, such as paraffin;(6) absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents.

Liquid dosage forms can include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the active ingredient, the liquid dosage forms may contain inertdiluents commonly used in the art, such as, for example, water or othersolvents, solubilizing agents and emulsifiers, such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (inparticular, cottonseed, groundnut, corn, germ, olive, castor and sesameoils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Ointments, pastes, creams and gels may contain, in addition to an activecompound, excipients, such as animal and vegetable fats, oils, waxes,paraffins, starch, tragacanth, cellulose derivatives, polyethyleneglycols, silicones, bentonites, silicic acid, talc and zinc oxide, ormixtures thereof.

Powders and sprays can contain, in addition to an active compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder, or mixtures of these substances.Sprays can additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any methods well known in the art of pharmacy. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, the particular mode of administration. The amount ofactive ingredient that can be combined with a carrier material toproduce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, out of onehundred percent, this amount will range from about 1 percent to aboutninety-nine percent of active ingredient, preferably from about 5percent to about 70 percent, most preferably from about 10 percent toabout 30 percent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions disclosed herein, such as dragees, capsules, pills andgranules, may optionally be scored or prepared with coatings and shells,such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions that can bedissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions that can be used include polymeric substances andwaxes. The active ingredient can also be in micro-encapsulated form, ifappropriate, with one or more of the above-described excipients.

Dosage forms for the topical or transdermal administration of a compoundof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. The active compound maybe mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants that may berequired.

The formulations disclosed herein can be delivered via a device.Exemplary devices include, but are not limited to, a catheter, wire,stent, or other intraluminal device. Further exemplary delivery devicesalso include a patch, bandage, mouthguard, or dental apparatus.Transdermal patches have the added advantage of providing controlleddelivery of a compound disclosed herein to the body. Such dosage formscan be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate of such flux can be controlled byeither providing a rate controlling membrane or dispersing the compoundin a polymer matrix or gel.

Ophthalmic formulations, eye ointments, drops, solutions and the like,are also contemplated as being within the scope of this invention.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which, in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions that are compatible with body tissue.

When the compounds disclosed herein are administered as pharmaceuticals,to humans and animals, they can be given per se or as a pharmaceuticalcomposition containing, for example, 0.1 to 99.5% (more preferably, 0.5to 90%) of active ingredient in combination with a pharmaceuticallyacceptable carrier.

The formulations can be administered topically, orally, transdermally,rectally, vaginally, parentally, intranasally, intrapulmonary,intraocularly, intravenously, intramuscularly, intraarterially,intrathecally, intracapsularly, intraorbitally, intracardiacly,intradermally, intraperitoneally, transtracheally, subcutaneously,subcuticularly, intraarticularly, subcapsularly, subarachnoidly,intraspinally, intrasternally or by inhalation.

One specific embodiment is an antitussive composition for peroraladministration comprising an agent that inhibits both a TRPC5-mediatedcurrent with an IC₅₀ of 1 micromolar or less, and an orally-acceptablepharmaceutical carrier in the form of an aqueous-based liquid, or soliddissolvable in the mouth, selected from the group consisting of syrup,elixer, suspension, spray, lozenge, chewable lozenge, powder, andchewable tablet. Such antitussive compositions can include one or moreadditional agents for treating cough, allergy or asthma symptom selectedfrom the group consisting of: antihistamines, 5-lipoxygenase inhibitors,leukotriene inhibitors, H3 inhibitors, β-adrenergic receptor agonists,xanthine derivatives, α-adrenergic receptor agonists, mast cellstabilizers, expectorants, NK1, NK2 and NK3 tachykinin receptorantagonists, and GABA_(B) agonists.

Still another embodiment is a metered dose aerosol dispenser containingan aerosol pharmaceutical composition for pulmonary or nasal deliverycomprising an agent that inhibits a TRPC5-mediated current with an IC₅₀of 1 micromolar or less. For instance, it can be a metered dose inhaler,a dry powder inhaler or an air-jet nebulizer.

Dosages

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound disclosed hereinemployed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compound employed, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the invention will bethat amount of the compound that is the lowest dose effective to producea therapeutic effect. Such an effective dose will generally depend uponthe factors described above. Generally, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisinvention for a patient will range from about 0.0001 to about 100 mg perkilogram of body weight per day. For example, the dose can be 0.1-50,0.1-25, 0.5-10, 1-10, or 5-10 mg/kg.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

Disease and Injury Models

Compounds that antagonize TRPC5 function may be useful in theprophylaxis and treatment of any of the foregoing injuries, diseases,disorders, or conditions. In addition to in vitro assays of the activityof these compounds, their efficacy can be readily tested in one or moreanimal models. By way of example, numerous well known animal modelsexist. One or more suitable animal models (e.g., suitable in light ofthe particular indication) can be selected.

Fear-related behaviors can be measured as described, e.g., in Riccio etal. Pain behaviors can be studied using various agents or procedures tosimulate pain resulting from injuries, diseases, or other conditions.Blackburn-Munro (2004) Trends in Pharmacological Sciences 25: 299-305(see, for example, Table 1). Behavioral characteristics of challengedanimals can then be observed. Compounds or procedures that may reducepain in the animals can be readily tested by observing behavioralcharacteristics of challenged animals in the presence versus the absenceof the test compound(s) or procedure.

Exemplary behavioral tests used to study chronic pain include tests ofspontaneous pain, allodynia, and hyperalgesia. Id. To assess spontaneouspain, posture, gait, nocifensive signs (e.g., paw licking, excessivegrooming, excessive exploratory behavior, guarding of the injured bodypart, and self-mutilation) can be observed. To measure evoked pain,behavioral responses can be examined following exposure to heat (e.g.,thermal injury model).

Exemplary animal models of pain include, but are not limited to, theChung model, the carageenan induced hyperalgesia model, the Freund'scomplete adjuvant induced hyperalgesia model, the thermal injury model,the formalin model and the Bennett Model. The Chung model of neuropathicpain (without inflammation) involves ligating one or more spinal nerves.Chung et al. (2004) Methods Mol Med 99: 35-45; Kim and Chung (1992) Pain50: 355-363. Ligation of the spinal nerves results in a variety ofbehavioral changes in the animals including heat hyperalgesia, coldallodynia, and ongoing pain. Compounds that antagonize TRPC5 can beadministered to ligated animals to assess whether they diminish theseligation-induced behavioral changes in comparison to that observed inthe absence of compound.

Useful anxiety and depression models include the maternal separationmodel, the elevated plus-maze model, the forced swim test, the tailsuspension test, the light/dark preference model, the light-enhancedstartle model, and the ultrasonic vocalization model.

Example 1 High Thoughput Screening Assay

The assay depended on detection of the rise in intracellular Ca²⁺concentration ([Ca²⁺]_(i)) following channel activation in cellsinducibly expressing the TRPC5 channel Ca²⁺ rise was quantified with theuse of fluorescent Ca²⁺ indicators that were loaded into cells andthereafter indicated the [Ca²⁺]_(i). Ca²⁺ influx followed activation ofthe TRPC5 channel. Compounds inhibiting the [Ca²⁺]_(i) rise wereconsidered hits for further investigation.

The commercially available HEK293/TREx line (Invitrogen) was stablytransfected with a TRPC5 construct and screened by conventional calciumimaging to find clones with TRPC5 expression following stimulation with1 μg/ml tetracycline. These cells were maintained in the growth mediumrecommended by the manufacturer supplemented with 100 μg/ml hygromycinto promote retention of the TRPC5 construct. After growing to nearconfluency, cells were plated at a density of ˜35,000 cells/well in 384well CellBind plates (Corning) in the presence of 1 μg/ml tetracycline,and allowed to grow for 20-30 hrs. A nearly confluent monolayerresulted. Cells were then loaded with Ca²⁺ dye: Fura-2/AM or Fluo4/AMwas added to the wells to a final concentration of 4 μM or 0.5 μM,respectively, and incubated for 80 min or 60 min, respectively, at roomtemperature. Supernatant was then removed from the cells by invertingplates with a sharp flick, and 40 μl Hank's Balanced Salt Solution(HBSS; 0.185 g/l D-glucose, 0.9767 g/l MgSO₄ (anhydrous), 0.4 g/l KCl,0.06 g/l KH₂PO₄ (anhydrous), 0.35 g/l NaHCO₃, 8.0 g/l NaCl, and 0.04788g/l Na₂HPO₄ (anhydrous); pH 7.4) was then added to each well. Following˜0.5 hour for recovery from loading, cells were assayed using theHamamatsu FDSS 6000 system, which permitted illumination alternately at340 nm and 380 nm for Fura-2 experiments, or at 485 nm for Fluo4experiments. Frames were acquired at a rate of 0.2 Hz. During the assay,the plates were continuously vortexed, with pipette mixing of wellsfollowing addition of each reagent. For the screening assay, 13 μl of adiluted compound stock (at 50 μM) was added to each well for 2 minutesfollowing the collection of a short (4 frame) baseline. 13 μl 62 mMhigh-Ca²⁺ Ringer solution (4.17 ml of normal ringer (with 2 mM Ca²⁺)plus 5.83 ml of isotonic calcium ringer (105 mM Ca²⁺; in this ringer allsodium has been replaced with calcium)) was then added to each well,achieving a final concentration of 14 mM Ca²⁺ and 10 μM test compound.Data was collected for ˜3 minutes following addition of high Ca²⁺Ringer, where the fluorescent intensity (for Fluo4) and the F340/F380ratio (for Fura-2) were proportional to the [Ca²⁺]_(i). Negativecontrols consisted of HEK293/TREx TRPC5 cells exposed to high Ca²⁺solution, but no compound. Positive control conditions consisted ofaddition of 2-APB, a promiscuous blocker of TRPC5 and other channels, tocolumns 23 and 24 of the plates, to a final concentration of 200 μM.These controls defined a screening window, and “hits” were defined asthose compounds inhibiting the fluorescence response by at least 40%.IC₅₀ values were determined for compounds defined as “hits.” The Fluo4cell-based fluorescence assay was used to determine the intracellularCa²⁺ concentration in the presence of varying drug concentration.Concentrations tested were 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM,and 0.625 μM. Compounds were tested in triplicate at all concentrations.Standard software was used to fit IC₅₀ curves.

Additionally or alternatively, efficacy can be represented as %inhibition in the presence (of a given concentration of compound) versusthe absence of compound or in comparison to a control compound. Forexample, efficacy can be represented as % inhibition of ion flux in thepresence versus the absence of compound.

Example 2 Patch Clamp Experiments

Patch clamp experiments permit the detection of currents through theTRPC5 channel in the cell line described above. In normal whole-cellpatch clamp recordings, a glass electrode is brought into contact with asingle cell and a high-resistance (gigaohm) seal is established with thecell membrane. The membrane is then ruptured to achieve the whole-cellconfiguration, permitting control of the voltage of the cell membraneand measurement of currents flowing across the membrane using theamplifier attached to the electrode and resulting in the replacement ofcytoplasm with the pipette solution. A perfusion system permits controlof the extracellular solution, including the addition of blockers andactivators of the current. The current can be activated by including 1.4μM free Ca²⁺ in the pipette (intracellular) solution, and 80 μM LaCl₃ inthe extracellular solution.

TRPC5 cells were induced 20-48 hours, removed from growth plates, andreplated at low density (to attain good single-cell physical separation)on glass coverslips for measurement. In some cases, cells were grown inlow density overnight on glass coverslips. Patch clamp recordings weremade in the whole-cell mode with a holding potential of −40 mV. Every 5seconds, a voltage ramp was applied from −120 to +100 mV, 400 ms induration. Currents elicited were quantified at −80 mV and +80 mV. Theinternal solution consisted of 140 mM cesium aspartate, 10 mM HEDTA, 2mM CaCl₂, 2.27 mM MgCl₂ and 10 mM HEPES, pH 7.2, with 1,400 nMcalculated free Ca²⁺. The external solution consisted of 150 mM NaCl,4.5 mM KCl, 3 mM MgCl₂, 10 mM HEPES, 10 mM glutamine, 1 mM EGTA, pH 7.4.Upon addition of LaCl₃, TRPC5 current was induced only inTRPC5-expressing cells and not in parental HEK293 TREx cells. Removal ofthe LaCl₃ stimulus causes most of the current to go away. Potentialblockers were tested for ability to block both inward and outwardcurrents in the continued presence of LaCl₃.

IC₅₀ of compounds was estimated by testing each compound at 5 μM and 500nM. When 5 μM compound showed no block, IC₅₀ was estimated as >10 μM.When 5 μM compound showed 50% or less block, a rough estimate of IC₅₀ inthe range of 5-10 μM could be made. IC₅₀ for compounds between 500 nMand 5 μM was similarly estimated. Compounds blocking 50% or more at 500nM are retested at multiple concentrations, and the % block at each isfitted by standard equations to determine IC₅₀ accurately using a 5-6point concentration/response experiment.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein, are hereby incorporatedby reference in their entirety as if each individual publication orpatent was specifically and individually indicated to be incorporated byreference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A method of treating anxiety in a subject, the methodcomprising administering an effective amount of a TRPC5 antagonist offormula (II), or a pharmaceutically acceptable salt thereof:

wherein R¹¹ is halo, C₁-C₆ alkoxy, cycyl, heterocyclyl, aryl, orheteroaryl; optionally substituted with 1-3 R¹⁶ _(;) R¹² is OR¹⁷, SR¹⁷,or NR¹⁴R¹⁵; R¹³ is H, cycyl, heterocyclyl, aryl, or heteroaryl;optionally substituted with 1-3 R¹⁸; each R¹⁴ and R¹⁵ is independently Hor C₁-C₆ alkyl optionally substituted with 1-3 R¹⁹; each R¹⁶ and R¹⁸ isindependently C₁-C₆ alkyl or C₁-C₆haloalkyl; R¹⁷ is C₁-C₆ alkyl,optionally substituted with 1-3 R¹⁹; R¹⁹ is C(O)OC₁-C₆alkyl,C(O)C₁-C₆alkyl, or OC(O)C₁-C₆alkyl.